Figure 4

Rescue and stability of the HAV constructs containing the EMCV IRES at the 5'NTR. (A) IF analysis of FRhK4 cells transfected with in vitro RNA transcripts from pHAVvec9, pHAVvec9-Bsd, or pHAV-IRES or mock-transfected. Cells were fixed with acetone two weeks post-transfection, and stained with anti-HAV neutralizing monoclonal antibodies K2-4F2 and K3-4C8 and FITC-conjugated goat anti-mouse antibodies. Micrographs were taken with a Zeiss microscope at 400× magnification. (B) Analysis of the stability of HAV recombinants containing the EMCV IRES. RT-PCR analysis of genomic RNA extracted form HAV/7, HAV-IRES, HAVvec9-Bsd virions and amplified using primers corresponding to nts 484-507 and 1167-1194 of HAV. As negative control, T7 polymerase in vitro transcripts from pHAV-IRES were spiked into media, layered on top of a 40% sucrose cushion, and sedimented by ultracentrifugation. RNA extracted from the pellet was used for the RT-PCR analysis (Naked-RNA control). The size of the DNA molecular weight markers (lane M) is indicated in bp.